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and comparison with cyclic nucleotide-based inhibitors for the regulatory subunits of PKA

Introduction

Due to the tetrameric composition of protein kinase A (PKA) of both, regulatory and catalytic subunits, inhibition of PKA can be achieved either by inhibitory analogues of cyclic AMP or by structures that block the ATP binding site (isoquinolines, staurosporine analogues). While inhibition of the regulatory subunits prevents the PKA holoenzyme from dissociation and liberation of free active regulatory subunits, a blockade of the catalytic ATP sites stops the phosphorylation process directly. Especially, membrane-permeant ATP site inhibitors, such as H 89, H 8, KT 5720 have become very popular in signal transduction studies, however, there are some serious limitations and potential pitfalls which should be known, in order to avoid drawbacks and disappointments:

Different Working Principle

Due to their different sites of action, ATP-site inhibitors and blockers of the regulatory subunits can differ in their effects on a given signaling pathway1. While H 89 and KT 5720 will interfere solely with the phosphorylation process of PKA, Rp-cAMPS and its analogues prevent the PKA holoenzyme from dissociation and thus act one step earlier. This can be an important difference, if the released regulatory subunits have special tasks on their own. Differences between e.g. H 89 and Rp-cAMPS and analogues were already reported:

Questionable Selectivity

Although the selectivity of ATP-site inhibitors of PKA, such as H 89 or KT 5720, seems to be rather good with respect to protein kinases C and G, this does obviously not hold true for other protein kinases. In addition, the usefulness of such compounds is at least questionable under the impression of numerous different types of ATP receptors in a cell, which are of course potential competitive binding sites as well. Thus it is not really astonishing, that more and more disturbing side effects are discovered, and that an increasing number of papers have already reported on that topic:

H 89 and KT 5720 lack specificity for PKA but inhibit other kinases with similar potency 20

H 89 blocks shaker-type K+-channels 22

H 89 inhibits Rho kinase23

H 89 has effects different from PKA inhibition24

In contrast to ATP, the number of cyclic AMP receptors is much more limited (PKA, PKG, PDE, CAP, CNG, EPAC/GEF), so a correspondingly selective inhibitor based on a cyclic nucleotide, such as Rp-cAMPS and its analogues would be much more focused on PKA, and thus have a clear advantage here.

Unclear Metabolic Fate

ATP-site directed inhibitors such as H 89 or KT 5720 are relatively complex organic compositions, whereas cyclic nucleotide analogues vary only slightly from natural cyclic AMP structure.Thus, the metabolic fate of H 89 or KT 5720 and the side effects of their metabolites in different tissues is more or less unknown, while cyclic nucleotide-based PKA inhibitors (Rp-cAMPS) are resistant against mammalian phosphodiesterases, the compulsory first step in metabolic degradation of these structures.

Conclusion

ATP-site directed inhibitors of PKA can be a useful additional control in concert with other tools, but should be used with caution and not be the single source of evidence for the involvement of PKA.